R01 Grant Abstract This proposal outlines a strategy for identifying the mechanisms that allow for precise specification of the stem cell fate within a dynamic tissue environment and for elucidating the mechanism of competition for stem cell niche occupancy. Accomplishing the aims in this proposal will be an important milestone toward the long-term goal of building a detailed model of epithelial stem cell regulation in order to develop regenerative medicine- based therapies and understand how the process fails in disease states such as cancer. The follicle stem cells (FSCs) have many features in common with mammalian epithelial progenitors and will likely be an excellent model for epithelial stem cell biology. Like several other types of epithelial stem cells in Drosophila and mammals, FSCs are stably maintained by a non-dedicated population of niche cells and depend on Wnt/wg and EGFR signals for self-renewal. In addition, FSCs produce daughter cells that differentiate over the course of several divisions but also retain the capacity to compete with resident stem cells for niche occupancy, much like transit amplifying cells in mammalian epithelial stem cell lineages. However, the precise mechanisms that govern these fundamental features of epithelial stem cell biology remain poorly understood. Based on published studies and significant preliminary data, the central hypothesis of the proposal is that FSC self-renewal is established by localized autocrine and juxtacrine signaling within the niche, and that a cellular memory of these niche signals preserves the ability of newly produced daughter cells to participate in niche competition. A corollary to this hypothesis is the idea that the genetic basis for niche competition is dependent on the relative capacity of a stem cell and a potential replacement cell for self-renewal or differentiation. The specific aims, which will provide complementary approaches to addressing these hypotheses, are: (1) determine how essential niche signals interact to precisely specify FSCs within a dynamic tissue; (2) identify the molecular switch that regulates the segregation of FSC and daughter cell fates; and (3) elucidate the mechanism of FSC niche competition. Newly developed tools and the finding that wg and EGFR are specific FSC niche signals make it possible to achieve the first aim. The second aim will be use biochemistry and cell biology techniques, and will build from the thorough understanding of Six-family transcription factors and groucho-mediated repression provided by the literature. The third aim will combine the detailed knowledge of FSC niche function provided by previous work and the first two aims with a new collection of hypercompetition mutants to obtain a comprehensive understanding of how niche competition is regulated. This project is significant because it will provide detailed mechanistic insight into the function of epithelial stem cell niches. It is an innovative departure from previous studies that focuses on building a comprehensive model of the FSC niche and on understanding novel concepts, the dynamic niche and niche competition. Ultimately, it will provide a detailed understanding of the conserved mechanisms that regulate epithelial stem cells.